PROJECT SUMMARY Herpes Simplex Virus Type 1 (HSV-1) is a common pathogen of the oral and genital mucosa with a seroprevalence last estimated at 54% in the United States. In cases of immune compromise, infection can have serious consequences including pneumonia, encephalitis, and keratitis. The large DNA virus employs a coordinated cascade of transcriptional events to efficiently generate progeny. The gene groups are temporally classified as immediate early (?), early (?), and late (?). DNA replication is required for efficient transcription of ? genes?this transcriptional strategy occurs in other virus families including adenoviridae, papillomaviridae, polyomaviridae, and baculoviridae. A better understanding of this essential transcriptional switch may aid in identification of novel drug targets and development of therapeutics. Recent work from our laboratory has demonstrated that initial rounds of DNA replication are sufficient for robust transcription of true late (?2) genes. Furthermore, viral DNA replication forks recruit numerous cellular factors including Mediator complex, Integrator complex, SUPT5H, SUPT6H, and RNA Polymerase II. We intend to expand on this observation and determine if DNA replication activates ?2 transcription at the level of initiation or elongation. We hypothesize that the act of DNA replication alters genome conformation facilitating recruitment of transcription factors, and that DNA synthesis components, in addition to the key viral regulatory proteins ICP4, and ICP27, recruit transcription factors that are important for late transcription. To assess the role of DNA replication in ?2 transcription we propose three aims. First, we will determine the extent of genome replication sufficient for activation of ?2 transcription and whether DNA replication promotes initiation or elongation. We will inhibit viral DNA replication at various time points and use RNA-Seq to assess the minimal amount of DNA replication sufficient for robust ?2 transcription. We will then determine the transcription defect for ?2 genes in the absence of DNA synthesis by performing ChIP-Seq. Transcription initiation will be assessed by analyzing the presence of initiation factors on ?2 promoters. Transcription elongation will be assessed by analyzing the occupancy of RNA polymerase II on ?2 transcription start sites and message bodies. Next, we will investigate whether DNA synthesis components play an additional role in ?2 transcription beyond the act of DNA replication. We will use temperature sensitive mutants in temperature shift experiments to determine if the component is required after initial rounds of DNA replication. Finally, we will elucidate the repertoire of cellular and viral factors required for robust ?2 transcription. Using aniPOND on prelabeled genomes we can determine what factors are bound to viral genomes immediately before and after DNA replication. Using HSV-1 mutants uniquely defective for ?2 transcription we can perform aniPOND to determine which of these factors are associated with ?2 transcription. The proposed study will elucidate the mechanism behind HSV-1 transcription control and how the virus hijacks cellular factors to accomplish this.